Fluorescence quenching with immunological pairs in immunoassays

ABSTRACT

Immunoassays are provided employing antibodies and a fluorescer-quencher (F-Q) chromophoric pair, wherein one or both of the chromophoric pair are bonded to antibodies. Depending on the particular ligand of interest, various reagent combinations can be employed, where the amount of quenching is directly related to the amount of ligand present in the assay medium. 
     In carrying out the assay, the unknown and antibody specific for the ligand of interest to which is bound one of the F-Q pair, are combined in an aqueous buffered medium. Depending on the protocol, different assay reagents are employed in the aqueous buffered medium: (1) ligand analog bonded to the other of the F-Q pair; (2) antibodies specific for the ligand to which is bound the other of the F-Q pair or; finally, (3) a combination of a plurality of ligands bonded together through linking groups to a hub molecule, usually a polymer, in combination with antibody bound to the other of the F-Q pair. The composition is irradiated with light at a wavelength, absorbed by the fluorescing molecule and the amount of fluorescence determined. By employing appropriate standards, the presence and amount of the ligand can be determined.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 591,386,filed June 30, 1975, now U.S. Pat. No. 3,996,345, which is acontinuation-in-part of application Ser. No. 497,167, filed Aug. 12,1974, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

There is a continuing need for rapid sensitive methods for determiningminute amounts of organic compounds. A number of techniques have beendeveloped toward this end. Among the commercially available techniquesare radioimmunoassay, spin-labeled immunoassay, for which reagents aresold under the trademark FRAT®, homogeneous enzyme immunoassay, forwhich reagents are sold under the trademark EMIT®, and hemagglutination(HI). These techniques are effective for determining amounts ofmaterials in the range of 10⁻⁶ to 10⁻¹⁰ M or less.

These techniques all involve the ability of a receptor molecule, usuallyan antibody, to be able to recognize a specific spatial and polarorganization of a molecule. Except for hemagglutination, the techniquesdepend upon providing a reagent which can compete with the moleculebeing assayed for the receptor. By being able to distinguish between thereagent which is bound to receptor and reagent which is unbound, one candetermine the amount of the compound of interest which is present.

In developing immunoassays, one is limited by the availability andproperties of an appropriate receptor. However, as for the otherreagents and the technique of measurement, there are a number ofdifferent considerations which make for a more accurate, convenient orcommercially desirable assay. First, it is desirable that there be aminimum number of measurements of the various reagents, as well astransfers of the various reagents. Secondly, the equipment for measuringshould be reasonably economical, so as to be accessible to a broad rangeof users. Thirdly, the reagents employed should be relatively stable, soas to be capable of storage and shipment. Fourthly, the method shouldnot be subject to significant interference from other materials whichmay be adventiously present in the sample to be assayed. Otherconsiderations are ease of training of technicans, absence of healthhazards, sensitivity, reproducibility, and applicability to a widevariety of ligands.

The subject invention is predicated on the phenomenon of energy transferbetween two chromophores. When a fluorescing chromophor is irradiatedwith light absorbed by the chromophore, the fluorescing chromophore candissipate the energy of the absorbed light by emitting light of longerwavelength, that is, fluorescing. If another chromophore is within lessthan 100A of the fluorescer and absorbs light at the wavelength ofemission, there is a probability, depending upon other factors, that thefluorescer will transfer to the other chromophore the energy which wouldotherwise have been emitted as light, in effect, quenching thefluoroescer.

2. Description of the Prior Art

U.S. Pat. No. 3,709,868 is exemplary of a radioimmunoassay. U.S. Pat.No. 3,690,834 is exemplary of a spin immunoassay. U.S. Pat. Nos.3,654,090 and 3,817,837 are exemplary of enzyme immunoassays. Articlesof interest include an article by Ludwig Brand and James R. Gohlke,entitled, Fluorescence Probes for Structure, Annual Review ofBiochemistry, 41, 843-868 (1972); and Stryer, Science, 162, 526 (1968).Also of interest is co-pending application Ser. No. 402,693, filed Oct.2, 1973.

SUMMARY OF THE INVENTION

A method is provided for determining the presence or amount of anorganic compound to which a receptor, usually antibody, is available orcan be prepared. The organic compound will be hereinafter referred to asa ligand.

In carrying out the assay, two chromophores are employed which are afluorescer-quencher pair. The amount of fluorescer within quenchingdistance of quencher is affected by the amount of ligand present in theassay medium.

One chromophore is introduced into the assay medium covalently bonded toa receptor composition which specifically binds to the ligand. Thesecond chromophore can be introduced into the assay medium in differentways: (1) covalently bonded to a receptor composition which is the sameor different from the receptor composition conjugated to the firstchromophore, but in both instances specifically binds to the ligand, andin the presence or absence of polyligand; or covalently bonded to ligandanalog, where the ligand analog can compete with ligand for the receptorcomposition. The choice of modes of introduction will depend to asignificant degree on the number of independent epitopic or haptenicsites present in the ligand.

Where the ligand has only one independent epitopic site (monoepitopic),usually one chromophore will be covalently bonded to a receptor forligand, and the other chromophore will be provided as covalently bondedto a ligand analog or a combination of poly(ligand analog) and thechromophore covalently bonded to receptor for ligand.

Where the ligand has a plurality of independent epitopic sites(polyepitopic), the modes indicated above may be used in addition to thefollowing modes. In one mode, the two chromophores are individuallybonded to receptor for ligand. In another mode, receptor for ligand isobtained from different species and one chromophore is bonded toreceptor for the ligand-receptor from one species and the otherchromophore bonded to receptor for ligand-receptor from the otherspecies. The latter method expands the versatility of the subject assayin allowing for common reagents for a wide variety of assays, simplifiespurification procedures, and allows for the determination of thepresence of assemblages, as distinct from the component parts.

The various materials are brought together in an aqueous bufferedmedium, incubated and irradiated with light absorbed by the fluorescermolecules. By determining the amount of fluorescence, after incubationfor a predetermined time interval or after the system has approachedequilibrium, and comparing the results obtained with one or more knownstandards, the presence or amount of ligand can be determined.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS Definitions

Ligand--an organic molecule or assemblage, normally greater than 100molecular weight and having at least one functionality, normally polar,for which a receptor is either naturally available or can be prepared.

Ligand analog--a mono- or polyvalent radical a substantial proportion ofwhich has the same spatial and polar organization as the ligand todefine one or more determinant or epitopic sites capable of competingwith the ligand for the binding sites of a receptor, and differs fromthe ligand in the absence of an atom or functional group at the site ofbinding to another molecule or in having a linking group which has beenintroduced in place of one or more atoms originally present in theligand. The ligand analog precursor is the compound employed forconjugating ligand or ligand analog to another molecule, e.g.,chromophore.

Assemblage--a combination of organic molecules bound together by otherthan covalent bonds, generally having molecular weights exceeding 600,usually exceeding 1,000 and may be 1,000,000 or more, for which receptoris either naturally available or can be prepared; an illustrativeassemblage is an antigen and antibody) or, a molecule prepared from twodiscrete entities, normally joined together by weak bonds, such as polarbonds or disulfide bonds, which under the conditions of the system arecapable of being in equilibrium with the individual entities.

Chromophore--a fluorescer or quencher molecule; in the subjectinvention, the fluorescer and quencher are interrelated. The fluorescermolecule is a chromophore which is able to absorb light at onewavelength and emit light at a longer wavelength. The quencher moleculeis capable of inhibiting fluorescence, when within a short distance,usually less than about 100 A, of the fluorescer molecule, by acceptingthe energy which would otherwise be emitted as fluorescent light. As faras the molecule or composition to which the chromophores are joined, inmost instances, the fluorescer and quencher will be interchangeable,although there will frequently be some preference. Therefore, forpurposes of generality, the two molecules will be referred to aschromophores, and individually referred to as Ch₁ and Ch₂.

Ligand analog-chromophore (ligand analog-(Ch₂)_(x))--ligand analog iscovalently bound to one or more fluorescent molecules or quenchermolecules. With small ligands, those below about 10,000 molecularweight, usually below about 2,000 molecular weight, the ligand analogwill usually be joined to fewer than 10 chromophores, usually from 1 to10 chromophores, not more than about 1 chromophore per 1,000 molecularweight. With a large ligand, at least 2,000 molecular weight, usually atleast about 10,000 molecular weight, a plurality of chromophores may becovalently bound to ligand analog. The number of chromophores presentwill be limited by the number which may be introduced without maskingtoo many epitopic sites of the ligand and the desire to have asufficient number of chromophores to insure a substantial amount ofquenching when receptor-Ch₁ is bound to the ligand analog-(Ch₂)_(x).

Poly(ligand analog)-poly(chromophore)[poly(ligandanalog)-poly(Ch₂)]--ligand analog and chromophore are bonded to a highmolecular weight (as compared to the ligand analog and chromophore)water soluble polyfunctionalized hub or nucleus molecule, to provide aplurality of ligand analog groups and chromophore groups spaced on thesurface of the molecule, so that when receptor-Ch₁ is bound to ligandanalog, some Ch₁ groups will be present within quenching distance of Ch₂groups.

Poly(ligand analog)--ligand analog groups are bonded to a high molecularweight (as compared to ligand analog) water soluble polyfunctionalizedhub or nucleus molecule, so that there are a sufficient number of ligandanalogs per unit area for quenching to occur when the poly(ligandanalog) is saturated with receptor-Ch₁ and receptor-Ch₂ in appropriateproportions.

Receptor-chromophore (receptor-Ch₁ and receptor-Ch₂)--a receptor is amolecule which is capable of distinguishing an epitopic site and bindingto such site. Usually receptors will have binding constants in excess of10⁴, frequently, in excess of 10⁶. For the most part, receptors areantibodies, although enzymes, nucleic acids, and certain globulins, mayalso act as receptors. In the subject invention, for the most part, thereceptors will be antibodies to which one or more, usually at least twoor more, chromophore groups will be bound.

Receptor composition--receptor composition is a homogeneous orheterogeneous composition capable of specific non-covalent binding toligand and ligand analog and includes anti-ligand (a composition whichspecifically recognizes the ligand) and a combination of anti-ligand andanti(anti-ligand) (a composition which specifically recognizes theanti-ligand).

GENERAL STATEMENT OF THE INVENTION

The method is predicated on the employment of two chromophores whichform a fluorescer-quencher pair. One of the chromophores is covalentlybonded to a composition (receptor) which specifically recognizes orbinds to a ligand. The other chromophore is covalently bonded to ligandanalog or receptor. When the two chromophore containing compositions areintroduced into the assay medium, the amount of ligand present in theassay solution will affect the amount of quencher within quenchingdistance of fluorescer. The assay may be carried out competitively,where ligand analog competes with ligand for receptor, ligand analogbeing present as poly(ligand analog) or covalently bonded tochromophore. The assay may also be carried out non-competitively withligands having a plurality of epitopic sites, where receptor having eachof the chromophores binds to ligand.

COMPOSITIONS

Depending upon the particular protocol employed and the ligand ofinterest, one or more of the following reagent compositions will beemployed in the assay medium: ligand analog-chromophore, poly(ligandanalog)-poly(chromophore), poly(ligand analog), one or two receptors andone or two receptor-chromophores. The first composition to be consideredwill be the ligand analog-chromophore.

Ligand Analog-Chromophore and Poly(Ligand Analog)-Poly(Chromophore)

The ligand analog-chromophore may be subdivided into two groups. Thefirst group is where the ligand analog-chromophore has a single ligandanalog and a single chromophore joined together by a relatively shortlinking group. In these instances, the ligand analog for the most partwill be haptenic, rather than antigenic, and generally be less thanabout 10,000 molecular weight, more usually less than about 6,000molecular weight, and frequently in the range of about 125 to 1,000molecular weight, excluding the linking group employed for linking tothe chromophore. For the most part, the ligand analog will differ fromthe ligand in having a particular functionality replaced by a bond, ahydrogen replaced by a bond, or a short carbon chain replaced by a bond(by bond, it is intended to include multiple bonds, as well as singlebonds) to join to the linking group for linking to the chromophore. Thevarious haptenic or low molecular weight ligands will be discussedsubsequently.

The linking group will normally have not more than about 10 atoms in thechain between the ligand and the chromophore, more usually have either abond or from about 1 to 6 atoms in the chain. The atoms for the mostpart will be carbon, oxygen, nitrogen and sulfur, particularly carbon,oxygen, and nitrogen.

The functionalities involved in the linking group will normally benon-oxo carbonyl (including imino and thionocarbonyl) oxy, amino(particularly tertiary amino or quaternary) or combinations thereof,e.g. amido, carbamyl, and amidino.

The two chromophores, either fluorescer or quencher, will normally haveeither an amino or alcohol function for reacting with a non-oxo carbonylfunction (including the nitrogen and sulfur analogs thereof) or have anon-oxo carbonyl function, which can be reacted with an amine or alcoholfunctionality.

Where the ligand is of at least 2,000 molecular weight, a plurality ofchromophore groups may be bound to the ligand. Usually, there will be atleast one chromophore group per 20,000 molecular weight, more usually atleast one chromophore group per 10,000 molecular weight and not morethan one chromophore group per 1,000 molecular weight, more usually notmore than one chromophore group per 2,000 molecular weight. Theconsiderations concerning the number of chromophores conjugated to theligand have been previously enumerated. The linking groups will be aspreviously described. Usually, the ligand will be an antigenicpolypeptide or protein having a plurality of amino groups. Activehalogen or non-oxo carbonyl (including nitrogen and sulfur analogs) canbe used for conjugation to form a covalent bond or amides, amidines,thionoamides, ureas, guanidines and thioureas.

Alternatively, the ligand and chromophore (Ch₁ or Ch₂) may be linked toa hub molecule (poly(ligand analog)-poly(chromophore). The hub moleculeor nucleus molecule can be employed with advantage for a variety ofreasons. The nucleus molecule will generally be a polymeric molecule ofrelatively high molecular weight, normally in excess of 20,000 molecularweight, frequently 60,000 molecular weight, and may be 10 million orhigher. The nucleus molecule will normally be water soluble ordispersible in an aqueous medium to provide a stable dispersion, wherethe dispersible material does not interfere with the absorption orirradiation of light. The nucleus molecule may be a naturally occurringmaterial, a modified naturally occurring material, or synthetic.Included among nucleus molecules are polypeptides, proteins,polysaccharides, synthetic polymers, and the like. The nature of the hubmolecule may be widely varied, so long as it is sufficientlyfunctionalized to permit the introduction of the ligand and thechromophore molecules.

Among proteins which can find use are albumins, globulins,proteoglycans, and the like; among polysaccharides are amylose,cellulose, agarose, dextrans, or the like, either as obtained orpartially degraded; among synthetic polymers, polyvinylalcohol,acrylates, copolymers thereof or the like may be employed.

Normally, there will be not less than about one conjugate (ligand analogor chromophore) molecule per 50,000 molecular weight, more usually notless than about one conjugate molecule per 25,000 molecular weight, andusually not more than about one conjugate molecule per 1,000 molecularweight, more usually not more than about one conjugate molecule per2,000 molecular weight.

The ratio of chromphore molecules to ligand will generally be from about0.05-20:1, more usually from about 0.5-20:1, preferably from about1-10:1, and more preferably from about 2-8:1.

Where the chromophore is the fluorescer molecule for the purposes ofthis invention, generally there will be at least about 0.5-20, moreusually from about 1-10, and preferably from about 2-7 fluorescingmolecules per ligand molecule. Where the chromophore is the quenchermolecule, the number of quencher molecules per ligand will generally befrom about 0.5-20, more usually from about 1-20, and preferably fromabout 2-15 per ligand molecule.

The conjugates to the hub molecule will have the same type of linkinggroup as was employed for joining the chromophore to the ligand. Theparticular choice of functionality will depend upon the availablefunctional groups on the nucleus molecule.

RECEPTOR-CHROMOPHORE

Since in most instances the receptor is antibody, the presentdescription will refer to antibody as exemplary of receptor. Antibodieshave a number of active amino groups which can be used for covalentlyconjugating the chromophore to the antibody. Conveniently, thechromophore can have a non-oxo carbonyl functionality (including thenitrogen and sulfur analogs thereof) or active α-halocarbonylfunctionality. Illustrative functionalities for linking the chromophoreto the antibody include acyl halides, mixed anhydrides, imidate alkylesters, isothiocyanate, chloro-, bromo- or iodoacetyl, and the like.

The conditions for conjugation employ moderate temperatures 0° to 40°C., in aqueous media at moderate pH. Conjugation of chromophores toprotein is known in the art. The, et al., Immunology, 18, 865 (1970);Cebra, et al., J. Immunol., 95, 230 (1965); Goldman, FluorescentAntibody Methods, Academic Press, New York (1968).

The number of chromophore groups which are conjugated to the antibodymay be varied over a relatively broad range, depending on thechromophore involved. There will be at least one chromophore group perantibody, and usually on the average, from about 2 to 30, more usuallyfrom about 3 to 25 chromophore groups per antibody. Where thechromophore is the fluorescer, the average number of chromophore groupsper antibody will be from about 1 to 20, usually 2 to 15 and moreusually 2 to 10. Where the chromophore is the quencher, the averagenumber of chromophore groups per antibody will be from about 2 to 30,usually 3 to 25, and more usually 5 to 25.

It should also be noted that when antibodies are prepared for a ligandhaving a plurality of epitopic sites, the receptor composition is nothomogeneous. That is, the receptor will have antibodies which recognizedifferent epitopic sites. In referring to receptor, it is intended toinclude all the antibodies which are capable of specifically binding toany of the epitopic sites of the ligand.

POLY(LIGAND ANALOG)

The poly(ligand analog) differs from the ligand analog-chromophore andpoly(ligand analog)-poly(chromophore) in that no chromophore is present,only ligand analog. The same types of nucleus molecules and the samedegree of conjugation apply for the poly(ligand analog) as for thepoly(ligand analog)-poly(chromophore). However, the ligand analog may bepresent in much higher ratio than the hub nucleus can accomodatereceptor. Therefore, while a minimum number of ligand analog groups areessential, the maximum number is one of expedience. The significantfactor is that receptor molecules when bound to poly(ligand analog) cancome sufficiently close to allow the chromophores to come withinquenching distance.

In choosing a nucleus molecule, a number of considerations will bear onthe choice. While it is not essential that the nucleus molecule be watersoluble, in most instances, it will be desirable. In any event, thenucleus molecule or composition will be capable of stable dispersion inan aqueous medium. Secondly, the nucleus molecule should not absorblight at the emission wavelength of the fluorescer to cause significantquenching. Thirdly, the nucleus molecule should not fluoresce at theemission wavelengths of the fluorescer when irradiated with the excitinglight. Therefore, any significant absorption by the nucleus moleculeshould be below about 520 nm, preferably below about 450 nm.

The nucleus molecule should be highly functionalized, preferably withamino or hydroxyl groups, although other reactive functionalities arealso useful, e.g. carboxy. Fourthly, the nucleus molecule should bestable under conditions of storage and use. Fifthly, the nucleusmolecule should be inert to functionalities present in the chromophoreand ligand, other than the functionality for linking. Finally, thenucleus molecule should not interfere with the immunoassay, for example,by having naturally occurring receptors which may be present inphysiological fluids which are studied.

While any size of molecule may be employed, very large molecules orcells will create practical problems. For example, a very large moleculepassing through the light beam of the fluorometer could provide a suddenincrease in the peak height. Therefore, the signal obtained would haveto be averaged over a reasonable period of time. Large molecules willalso result in increased scatter, but the scatter could be compensatedfor by an appropriate optical system. Preferably, for the most part,molecules will be employed which are less than about 10 millionmolecular weight, more preferably from about 30,000 to 1,000,000molecular weight.

CHROMOPHORE

Since antibodies are normally present in the assay medium, and proteinsabsorb light of wavelengths up to about 310nm, the fluorescer will havesubstantial absorption higher than 310 nm, normally higher than 350 nm,and preferably higher than about 400 nm. The choice of fluorescer willalso be governed by the particular ligand of interest. The fluorescershould absorb light at a higher wavelength than the ligand or ligandanalog of interest. A high extinction co-efficient is desirable, greatlyin excess of 10, preferably in excess of 10³, and particularly preferredin excess of 10⁴. A good quantum yield should be available in theaqueous medium for the fluorescer. As a matter of convenience, theabsorption peak of the fluorescer should not vary significantly withvariation in the ligand.

A number of different fluorescers are described in the articlespreviously noted; namely, Stryer, supra, and Brand, et al., supra.

One group of fluorescers having a number of the desirable propertiesdescribed previously are the xanthene dyes, which include thefluoresceins derived from 3,6-dihydroxy-9-phenyl-xanthhydrol androsamines and rhodamines, derived from 3,6-diamino-9-phenylxanthhydrol.The rhodamines and fluoresceins have a 9-o-carboxyphenyl group, and arederivatives of 9-o-carboxyphenylxanthhydrol.

These compounds are commercially available with substituents on thephenyl group which can be used as the site for bonding or as the bondingfunctionality. For example, amino and isothiocyanate substitutedfluorescein compounds are available.

Another group of fluorescent compounds are the naphthylamines, having anamino group in the alpha or beta position, usually alpha position.Included among the naphthylamino compounds are1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonateand 2-p-toluidinyl-6-naphthalene sulfonate.

Other dyes include 3-phenyl-7-isocyanatocoumarin, acridines, such as9-isothiocyanatoacridine and acridine orange;N-(p-(2-benzoxazolyl)phenyl)maleimide; benzoxadiazoles, such as4-chloro-7-nitrobenzo-2-oxa-1,3-diazole and7-(p-methoxybenzylamino)-4-nitrobenzo-2-oxa-1,3-diazole; stilbenes, suchas 4-dimethylamino-4'-isothiocyanatostilbene and4-dimethylamino-4'-maleimidostilbene; N,N'-dioctadecyloxacarbocyaninep-toluenesulfonate; pyrenes, such as 8-hydroxy-1,3,6-pyrenetrisulfonicacid, and 1-pyrenebutyric acid, merocyanine 540, rose bengal,2,4-diphenyl-3(2H)-furanone, as well as other readily availablefluorescing molecules. These dyes, either have active functionalities orsuch functionalities may be readily introduced.

Similar considerations involved with the fluorescer molecule areapplicable to the quenching molecule, except that a good fluorescentquantum yield is not required where fluorescence of the fluorescer isbeing measured. An additional consideration for the quenching moleculeis that it has its absorption at an emission wavelength of thefluorescer. Good overlap of the fluorescer emission and quencherabsorption is desirable.

It should be noted that both the absorption and emission characteristicsof the dye may vary from being free in solution and being bound to aprotein or ligand. Therefore, when referring to the various ranges andcharacteristics of the dyes, it is intended to indicate the dye asemployed and not the dye which is unconjugated and characterized in anarbitrary solvent. In the area of overlap between fluorescence andquenching, the quencher should have extinction coefficients of the sameorder or higher than those set forth for absorption by the fluorscingmolecule.

LIGAND

As indicated, the ligand will vary widely, normally having a molecularweight of at least 110, more usually at least 125 with the maximummolecular weight unlimited, although usually not exceeding 10 million.For the most part, the significant factor concerning a ligand is that areceptor can be made to the ligand or is available. Normally, receptorscan be made for most organic compounds having a polar functionality.Compounds for which antibodies can be formed by bonding the compound toa compound having antigenic properties are referred to as haptens. Thosecompounds which elicit antibody formation without chemical modificationare referred to as antigens. See Kabat, et al., ExperimentalImmunochemistry, Charles C. Thomas, Springfield, Illinois, 1967.

The non-polymeric ligands of interest will normally be of from about 125to 2,000 molecular weight. These compounds involve a wide variety ofcompounds of varying structure, functionality, and physiologicalproperties. The compounds may be acyclic, alicyclic or heterocyclic,both mono- and polycyclic. The heteroatoms involved include oxygen,nitrogen, sulfur, halogen (fluorine, chloride, bromine and iodine)boron, phosphorous, metal cations of Groups 1A and 2A of the PeriodicChart, transition metals, and the like.

The functionalities include alcohols, ethers, carboxylic acids, estersand amides, amines (primary, secondary, tertiary and quaternary) halo,nitrilo, mercapto, and the like. Normally, the compounds will becomposed solely of carbon, hydrogen, oxygen, nitrogen, halogen andphosphorous, particularly carbon, hydrogen, oxygen, and nitrogen andwhere salts are involved, the appropriate metal counterion or ammoniumcounterion.

Heterocyclic rings which are present include pyrrole, pyridine,piperidine, indole, thiazole, piperazine, pyran, coumarin, pyrimidine,purine, triazine, imidazole, and the like.

Because of the wide variety of compounds which can be determined inaccordance with the subject assay, the different groups will be brokendown into various, frequently artificial, categories, either by thepresence of a particular functionality or ring structure, or because ofsharing a particular function or because of being recognized as a class.

The first class of compounds of interest are those having an aminogroup, either as a heterocyclic member, or as a functionality on analiphatic chain. These compounds will normally be of from about 110 to800 molecular weight, more usually of about 125 to 650 molecular weight.These compounds frequently have an amino group separated by 2 to 3aliphatic carbon atoms from a benzene ring.

The first group of compounds of interest are the alkaloids and themetabolites of those alkaloids which are ingested. The first group ofimportant alkaloids are alkaloids of the morphine group. Included inthis group are morphine, codeine, heroin, morphine glucuronide and thelike.

The next group of alkaloids are the cocaine alkaloids, which includes,particularly as metabolites, benzoyl ecgonine and ecgonine.

Another group of alkaloids are the cinchona alkaloids which includesquinine.

The isoquinoline group of alkaloids includes mescaline.

The benzylisoquinoline alkaloid group includes papaverine.

The phthalide isoquinoline alkaloid group includes narcotine, narceine,and cotarnine.

The indolopyridocoline alkaloid group includes yohimbine and reserpine.

The ergot alkaloid group includes ergotamine and lysergic acid.

Other groups of alkaloids are strychnine alkaloids, pyridine alkaloids,piperidine alkaloids, pyrrolizidine alkaloids, and the like.

The alkaloids of primary interest are those which come within thecategory of drugs of abuse, such as morphine, cocaine, mescaline, andlysergic acid, which may be analyzed for the compound or its metabolite,depending on the physiological fluid which is analyzed for its presence.

A number of synthetic drugs mimic the physiological properties, in partor in whole, of the naturally occurring drugs of abuse. Included amongthese drugs are methadone, meperidine, amphetamine, methamphetamine,glutethimide, diphenylhydantoin, and drugs which come within thecategory of benzdiazocycloheptanes, phenothiazines and barbiturates.

Drugs of interest because of their physiological properties are thosewhich are referred to as catecholamines. Among the catecholamines areepinephrine, ephedrine, L-dopa, and norepinephrine.

Other drugs of interest are the tranquilizer Meprobamate, Tegritol andsuccinimides, such as Ethoxsumide.

Other compounds of interest are tetrahydrocannabinol, cannabinol, andderivatives thereof, primarily compounds derived from marijuana,synthetic modifications and metabolites thereof.

Another group of compounds of significant interest are the steroids. Thesteroids include estrogens, gestogens, androgens, adrenocorticalhormones, bile acids, cardiotonic glycoids, algycones, saponins andsapogenins.

Another class of compounds are the vitamins, such as vitamin A, the Bgroup, e.g. vitamin B₁, B₆, and B₁₂, E, K, and the like.

Another class of compounds are the sugars, both the mono- andpolysaccharides, particularly di- and higher order polysaccharides.

Another class of compounds is the prostaglandins.

Another class of compounds are the amino acids, polypeptides andproteins. Polypeptides usually encompass from about 2 to 100 amino acidunits (usually less than about 12,000 molecular weight). Largerpolypeptides are arbitrarily called proteins and are usually composed offrom about 1 to 20 polypeptide chains. Poly(amino acid) will be used asgeneric to polypeptides and proteins. Of particular interest among aminoacids is thyronines, both the tri- and tetraiodo. The poly(amino acid)semployed in this invention employing two antibodies as reagents willgenerally range from about 5,000 to 10⁷, usually 10⁴ to 10⁶ molecularweight. Of particular interest among polypeptides and proteins[poly(amino acids)] are hormones, globulins, antigens and compositionsfound to have specific physiological activities.

The wide variety of proteins may be considered as to the family ofproteins having similar structural features, proteins having particularbiological functions, proteins related to specific microorganisms,particularly disease causing microorganisms, etc.

The following are classes of proteins related by structure:

protamines

histones

albumins

globulins

scleroproteins

phosphoproteins

mucoproteins

chromoproteins

lipoproteins

nucleoproteins

unclassified proteins, e.g. somalotropin, prolactin, insulin, pepsin.

A number of proteins found in the human plasma are important clinicallyand includes:

Prealbumin

Albumin

α₁ -Lipoprotein

α₁ -Acid glycoprotein

α₁ -Antitrypsin

α₁ -Glycoprotein

Transcortin

4.6S-Postalbumin

Tryptophan-poor

α₁ -glycoprotein

α₁ χ-Glycoprotein

Thyroxin-binding globulin

Inter-α-trypsin-inhibitor

Gc-globulin:

(Gc 1-1),

(Gc 2-1),

(Gc 2-2),

Haptoglobin:

(Hp 1-1),

(Hp 2-1),

(Hp 2-2),

Ceruloplasmin

Cholinesterase

α₂ -Lipoprotein(s)

α₂ -Macroglobulin

α₂ -HS-glycoprotein

Zn-α₂ -glycoprotein

α₂ -Neuramino-glycoprotein

Erythropoietin

β-lipoprotein

Transferrin

Hemopexin

Fibrinogen

Plasminogen

β₂ -glycoprotein I

β₂ -glycoprotein II

Immunoglobulin G

(IgG) or γG-globulin

Mol. formula:

γ₂ κ₂ or γ₂ λ₂

Immunoglobulin A (IgA)

or γA-globulin

Mol. formula:

(α₂ κ₂)^(n) or (α₂ λ₂)^(n)

Immunoglobulin M

(IgM) or γM-globulin

Mol. formula:

(μ₂ κ₂)⁵ or (μ₂ λ₂)⁵

Immunoglobulin D(IgD)

or γD-Globulin (γD)

Mol. formula:

(δ₂ κ₂) or (δ₂ λ₂)

Immunoglobulin E (IgE)

or γE-Globulin (γE)

Mol. formula:

(ε₂ κ₂) or (ε₂ λ₂)

Free light chains

Complement factors:

C'1

C'1q

C'1r

C'1s

C'2

C'3

β₁ A

α₂ D

C'4

C'5

C'6

C'7

C'8

C'9.

Important blood clotting factors include:

                  TABLE VII                                                       ______________________________________                                        CLOTTING FACTORS                                                              International designation                                                                        Name                                                       ______________________________________                                        I               Fibrinogen                                                    II              Prothrombin                                                   IIa             Thrombin                                                      III             Tissue thromboplastin                                         V and VI        Proaccelerin, accelerator                                                     globulin                                                      VII             Proconvertin                                                  VIII            Antihemophilic globulin (AHG)                                 IX              Christmas factor,                                                             plasma thromboplastin                                                         component (PTC)                                               X               Stuart-Prower factor,                                                         autoprothrombin III                                           XI              Plasma thromboplastin                                                         antecedent (PTA)                                              XII             Hagemann factor                                               XIII            Fibrin-stabilizing factor                                     ______________________________________                                    

Important protein hormones include:

Peptide and Protein Hormones

Parathyroid hormone

(parathormone)

Thyrocalcitonin

Insulin

Glucagon

Relaxin

Erythropoietin

Melanotropin

(melanocyte-stimulating hormone; intermedin)

Somatotropin

(growth hormone)

Corticotropin

(adrenocorticotropic hormone)

Thyrotropin

Follicle-stimulating hormone

Luteinizing hormone

(interstitial cell-stimulating hormone)

Luteomammotropic hormone

(luteotropin, prolactin)

Gonadotropin

(chorionic gonadotropin).

Tissue Hormones

Secretin

Gastrin

Angiotensin I and II

Bradykinin

Human placental lactogen

Peptide Hormones from the Neurohypophysis

Oxytocin

Vasopressin

Releasing factors (RF)

CRF, LRF, TRF, Somatotropin-RF, GRF, FSH-RF, PIF, MIF.

Other polymeric materials of interest are mucopolysaccharides andpolysaccharides.

Illustrative antigenic polysaccharides derived from microorganisms areas follows:

    ______________________________________                                        Species of Microorganisms                                                                       Hemosensitin Found in                                       ______________________________________                                        Streptococcus pyogenes                                                                          Polysaccharide                                              Oiplococcus pneumoniae                                                                          Polysaccharide                                              Neisseria meningitidis                                                                          Polysaccharide                                              Neisseria gonorrhoeae                                                                           Polysaccharide                                              Corynebacterium diphtheriae                                                                     Polysaccharide                                              Actinobacillus mallei;                                                                          Crude extract                                                Actinobacillus whitemori                                                     Francisella tularensis                                                                          Lipopolysaccharide                                                            Polysaccharide                                              Pasteurella pestis                                                            Pasteurella pestis                                                                              Polysaccharide                                              Pasteurella multocida                                                                           Capsular antigen                                            Brucella abortus  Crude extract                                               Haemophilus influenzae                                                                          Polysaccharide                                              Haemophilus pertussis                                                                           Crude                                                       Treponema reiteri Polysaccharide                                              Veillonella       Lipopolysaccharide                                          Erysipelothrix    Polysaccharide                                              Listeria monocytogenes                                                                          Polysaccharide                                              Chromobacterium   Lipopolysaccharide                                          Myobacterium tuberculosis                                                                       Saline extract of 90%                                                          phenol extracted                                                              mycobacteria and poly-                                                        saccharide fraction of                                                        cells and tuberculin                                       Klebsiella aerogenes                                                                            Polysaccharide                                              Klebsiella cloacae                                                                              Polysaccharide                                              Salmonella typhosa                                                                              Lipopolysaccharide,                                                            Polysaccharide                                             Salmonella typhi-murium;                                                                        Polysaccharide                                               Salmonella derby                                                              Salmonella pullorum                                                          Shigella dysenteriae                                                                            Polysaccharide                                              Shigella flexneri                                                             Shigella sonnei   Crude, polysaccharide                                       Rickettsiae       Crude extract                                               Candida albicans  Polysaccharide                                              Entamoeba histolytica                                                                           Crude extract                                               ______________________________________                                    

Another group of compounds are the antibiotics such as penicillin,actinomycin, chloromycetin, and the like.

Individual compounds of interest are serotonin, spermine, andphenylpyruvic acid.

Finally, compounds which are pesticides, such as fungicides,insecticides, bactericides, and nematocides, may also be of interest forassaying.

Other than compounds of interest, cells, viruses, and other biologicalaggregations which are antigenic or to which naturally occurringreceptors can be found may also be assayed for.

The microorganisms which are assayed may be intact, lysed, ground orotherwise fragmented, and the resulting composition or portion, e.g. byextraction, assayed. Microorganisms of interest include:

    ______________________________________                                        Corynebacteria                                                                 Corynebacterium diptheriae                                                   Pneumococci                                                                    Diplococcus pneumoniae                                                       Streptococci                                                                   Streptococcus pyogenes                                                        Streptococcus salivarus                                                      Staphylococci                                                                  Staphylococcus aureus                                                         Staphylococcus albus                                                         Neisseriae                                                                     Neisseria meningitidis                                                        Neisseria gonorrheae                                                         Enterobacteriaciae                                                             Escherichia coli                                                              Aerobacter aerogenes                                                                              The coliform bacteria                                     Klebsiella pneumoniae                                                         Salmonella typhosa                                                            Salmonella choleraesuis                                                                           The Salmonellae                                           Salmonella typhimurium                                                        Shigella dysenteriae                                                          Shigella schmitzii                                                            Shigella arabinotarda                                                                             The Shigellae                                             Shigella Flexneri                                                             Shigella boydii                                                               Shigella Sonnei                                                              Other enteric bacilli                                                          Proteus vulgaris                                                              Proteus mirabilis   Proteus species                                           Proteus morgani                                                               Pseudomonas aeruginosa                                                        Alcaligenes faecalis                                                          Vibrio cholerae                                                              Hemophilus-Bordetella group                                                    Hemophilus influenzae,                                                                        H. ducreyi                                                                    H. hemophilus                                                                 H. aegypticus                                                                 H. paraiufluenzae                                             Bordetalla pertussis                                                         Pasteurellae                                                                   Pasteurella pestis                                                            Pasteurella tulareusis                                                       Brucellae                                                                      Brucella melitensis                                                           Brucella abortus                                                              Brucella suis                                                                Aerobic Spore-forming Bacilli                                                  Bacillus anthracis                                                            Bacillus subtilis                                                             Bacillus megaterium                                                           Bacillus cereus                                                              Anaerobic Spore-forming Bacilli                                                Clostridium botulinum                                                         Clostridium tetani                                                            Clostridium perfringens                                                       Clostridium novyi                                                             Clostridium septicum                                                          Clostridium histolyticum                                                      Clostridium tertium                                                           Clostridium bifermentans                                                      Clostridium sporogenes                                                       Mycobacteria                                                                   Mycobacterium tuberculosis hominis                                            Mycobacterium bovis                                                           Mycobacterium avium                                                           Mycobacterium leprae                                                          Mycobacterium paratuberculosis                                               Actinomycetes  (fungus-like bacteria)                                          Actinomyces israelii                                                          Actinomyces bovis                                                             Actinomyces naeslundii                                                        Nocardia asteroides                                                           Nocardia brasiliensis                                                        The Spirochetes                                                                Treponema pallidum                                                                            Spirillum minus                                               Treponema pertenue                                                                            Streptobacillus moniliformis                                  Treponema carateum                                                            Borrelia recurrentis                                                          Leptuspira                                                                     icterohemorrhagiae                                                           Leptospira canicola                                                          Mycoplasmas                                                                    Mycoplasma pneumoniae                                                        Other pathogens                                                                Listeria monocytogenes                                                        Erysipelotrix rhusiopathiae                                                   Streptobacillus moniliformis                                                  Donvania granulomatis                                                         Bartonella bacilliformis                                                     Rickettsiae (bacteria-like parasites)                                         Rickettsia prowazekii                                                         Rickettsia mooseri                                                            Rickettsia rickettsii                                                         Rickettsia conori                                                             Rickettsia australis                                                          Rickettsia sibiricus                                                          Rickettsia akari                                                              Rickettsia tsutsugamushi                                                      Rickettsia burnetii                                                           Rickettsia quintana                                                           Chlamydia (unclassifiable parasites bacterial/viral)                           Chlamydia agents (naming uncertain)                                          Fungi                                                                         Cryptococcus neoformans                                                       Blastomyces dermatidis                                                        Histoplasma capsulatum                                                        Coccidioides immitis                                                          Paracoccidioides brasiliensis                                                 Candida albicans                                                              Aspergillus fumigatus                                                         Mucor corymbifer (Absidia corymbifera)                                        Rhizopus oryzae                                                               Rhizopus arrhizus  Phycomycetes                                               Rhizopus nigricans                                                            Sporotrichum schenkii                                                         Fonsecaea pedrosoi                                                            Fonsecaea compacta                                                            Fonsecaea dermatitidis                                                        Cladosporium carrionii                                                        Phialophora verrucosa                                                         Aspergillus nidulans                                                          Madurella mycetomi                                                            Madurella grisea                                                              Allescheria boydii                                                            Phialosphora jeanselmei                                                       Microsporum gypseum                                                           Trichophyton mentagrophytes                                                   Keratinomyces ajelloi                                                         Microsporum canis                                                             Trichophyton rubrum                                                           Microsporum andouini                                                          Viruses                                                                       Adenoviruses                                                                  Herpes viruses                                                                 Herpes simplex                                                                Varicella (Chicken pox)                                                       Herpes Zoster (Shingles)                                                      Virus B                                                                       Cytomegalovirus                                                              Pox Viruses                                                                    Variola (smallpox)                                                            Vaccinia                                                                      Poxvirus bovis                                                                Paravaccinia                                                                  Molluscum contagiosum                                                        Picornaviruses                                                                 Poliovirus                                                                    Coxsackievirus                                                                Echoviruses                                                                   Rhinoviruses                                                                 Myxoviruses                                                                    Influenza (A, B, and C)                                                       Parainfluenza (1-4)                                                           Mumps Virus                                                                   Newcastle Disease Virus                                                       Measles Virus                                                                 Rinderpest Virus                                                              Canine Distemper Virus                                                        Respiratory Syncytial Virus                                                   Rubella Virus                                                                Arboviruses                                                                    Eastern Equine Eucephalitis Virus                                             Western Equine Eucephalitis Virus                                             Sindbis Virus                                                                 Chikungunya Virus                                                             Semliki Forest Virus                                                          Mayora Virus                                                                  St. Louis Encephalitis Virus                                                  California Encephalitis Virus                                                 Colorado Tick Fever Virus                                                     Yellow Fever Virus                                                            Dengue Virus                                                                 Reoviruses                                                                     Reovirus Types 1-3                                                           Hepatitis                                                                      Hepatitis A Virus                                                             Hepatitis B Virus                                                            Tumor Viruses                                                                 Rauscher Leukemia Virus                                                       Gross Virus                                                                   Maloney Leukemia Virus                                                        Friend Leukemia Virus                                                         Mouse Mammary Tumor Virus                                                     Avian Leucosis Virus                                                          Rous Sarcoma Virus                                                            Polyoma Virus                                                                 Simian Virus 40                                                               Papilloma Virus                                                               Preparations of microorganisms include:                                       Streptococcus pyogenes, protein                                               Pasteurella pestis, protein toxin                                             Clostridium tetani, toxoid                                                    Clostridium perfringens, α-lecithinase                                  Escherichia coli, filtrates                                                   Treponema reiteri, protein extract                                            Corynebacterium diphtheriae, toxin, toxoid                                    Myobacterium tuberculosis, protein                                            M. tuberculosis, cytoplasm                                                    M. tuberculosis, culture filtrate and tuberculin                              Mycoplasma pneumoniae, "crude" antigen                                        ______________________________________                                    

IMMUNOASSAY

The subject immunoassays are based on the degree of quenching occurringin a solution where fluorescent molecules are irradiated with lightabsorbed by the fluorescer, preferably within the absorption peak, as afunction of the amount of ligand in the medium. Thus, the number offluorescer and quencher molecules which are brought together to within adistance where quenching can occur in related to the amount of ligandpresent in the assay medium.

The assay can be carried out with receptors for the ligand (anti-ligand)conjugated to the chromophore (antiligand)-chromophore or receptor forthe anti-ligand (anti(anti-ligand)) conjugated to the chromophore(anti(anti-ligand)-chromophore). For reasons which will be discussedsubsequently, the latter technique (the double receptor technique)provides procedural advantages, as well as providing assay capabilitiesnot available with the single receptor technique. The double receptortechnique binds receptor-chromophore indirectly to the ligand through areceptor (anti-ligand) intermediary, which now allows for an additionaldegree of freedom in varying the reagents.

In carrying out the assay employing the single receptor technique, theligand analog reagent has ligand analog bound either directly(covalently) to a chromophore, ligand analog-(Ch₂)_(x) or poly(ligandanalog)-poly(Ch₂), or indirectly (through receptor-Ch₂) to a chromophore(Ch₂). The assay is then carried out by combining in the assay medium,the ligand bound to Ch₂, receptor-Ch₁, and the unknown. Various ordersof addition are permissible. Where ligand analog is to be boundindirectly to Ch₂, receptor-Ch₁ and receptor-Ch₂, may be added stepwiseor substantially simultaneously.

Conveniently, the receptor-Ch₁ and receptor-Ch₂ may be combined togetheras a single reagent at the proper ratio. In this manner, the ratio ofthe two common receptors can be carefully controlled and accuratelyadded to the assay mixture. The mixture can be a dry lyophilized mixtureor an aqueous, normally buffered (pH 5-10; usually 6.5-8.5) solution ofany desired concentration.

The concentration of ligand of interest will generally range from about10⁻⁴ to 10⁻¹⁴, more usually from about 10⁻⁶ to 10⁻¹² M, most usually10⁻⁶ to 10⁻¹⁰ M. The concentrations of reagents will reflect theconcentration of interest of the ligand.

The medium will normally be aqueous, having from 0 to 20, more usuallyfrom 0 to 10 volume percent of a polar organic solvent. Illustrativepolar orlganic solvents include ethylene glycol, ethanol, carbitol,dimethylformamide, dimethylsulfoxide and the like. Preferably, theaqueous medium will be substantially free of other polar solvents. Themedium will normally be buffered in the range of about 5 to 10,preferably from about 6.5 to 8.5, and more preferred from about 7 to8.5. Various buffers may be used, such as borate, phosphate, carbonate,barbituric acid, tris, and the like. The particular buffer employed isnot critical to this invention, but in particular assays, one buffer maybe preferred over another. The buffer concentration will normally rangefrom about 0.005 M to 0.5 M, more usually from about 0.01 M to about 0.1M.

During the assay, moderate temperatures normally will be employed,generally ranging from about 0° C. to 45° C., more usually ranging fromabout 15° C. to 40° C. The particular temperature chosen will depend onconvenience, and on the effect of temperature on fluorescenceefficiency, and on the binding constant of the receptor to the ligand.The assay performance will be improved at lower temperatures, since bothfluorescence efficiency and binding constants are enhanced.

For convenience, the single receptor assays will be divided into thosewhere ligand is bound convalently to chromophore and those where ligandis bound indirectly through receptor to chromophore.

The first assay to be considered will be with those compositions wherechromophore is covalently bound to ligand. As previously indicated, asingle chromophore may be bound to a single ligand or by employing anucleus molecule, a plurality of ligands may be bound to a plurality ofchromophore groups. Alternatively, with large ligands such as proteins,a plurality of chromophore groups may be bound to the ligand.

The ligand analog-chromophore will generally be at a concentration notgreater than 100 times the highest concentration and not less than 0.01times the lowest concentration of the concentration range of interest,more usually being in the range from the highest concentration ofinterest to not less than 0.1 times the lowest concentration ofinterest, and preferably within an order of magnitude or a factor of 10of the lowest concentration of interest. The receptor-chromophoreconcentration is then determined by adding a sufficient amount of thereceptor to obtain at least 10 percent quenching, preferably at least 20percent quenching, and up to 100 percent quenching, usually from about20 to 80 percent quenching, and preferably from about 50 to 80 percentquenching. The amount of receptor-chromophore employed will be relatedto the binding constant, the concentration of interest which affects theconcentration of the ligand-chromophore, the sensitivity of theinstrument, and the like.

While the chromophore bound to the ligand may be quencher, for the mostpart, the chromophore bound to ligand will be fluorescer. This is not amatter of operability, but rather expedience. In most cases, thereceptor is antibody, which will be a complex protein mixture,containing antibody for the ligand, as well as other antibodies andproteins. When the antibody composition is labeled with chorophore, asubstantial proportion of the chromophore will be bound to protein otherthan the antibody for the ligand (anti-ligand). Therefore, if fluorescerwas bound to receptor, this would result in a large backgroundfluorescence in the assay medium. Alternatively, when a relatively puresample of anti-ligand is available, the preferred procedure would be tobind ligand to quencher, rather than fluorescer.

The particular order of addition of the various materials to the assaymedium is not critical to this invention. The unknown and ligandanalog-chromophore may be combined simultaneously withreceptor-chromophore or the materials added sequentially. Preferably,the unknown is combined with the receptor-chromophore and incubated fora sufficient time, so as to approach equilibrium. Therefore, theavailable binding sites of the receptor are reduced in proportion to theamount of unknown present in the assay medium. The ligandanalog-chromophore may then be added and incubated and the solution thentransferred to a fluorometer and the fluorescence intensity determinedon exciting with light at a wavelength or wavelengths absorbed by thefluorescer.

Incubation times will be dependent upon the temperature employed, thebinding constant of the receptor and the concentrations of the materialspresent in the assay medium. Normally, incubation times will be at leastabout 5 sec and preferably not exceeding about 6 hours, more usuallybeing in the range of about 30 sec to 2 hours, preferably, 1 to 30 min.Temperatures of incubation will generally vary from about 15° to 40° C.

By employing a series of solutions having known concentrations ofligand, one can provide a standard curve relating fluorescence orpercent quenching to concentration of ligand. The fluorescence resultingfrom an assay medium with an unknown can then be directly related to theconcentration of the unknown in the assay medium.

In a second mode, in which ligand is bound indirectly to a chromophore,the anti-ligand is divided into two parts and one part conjugated withfluorescer and the other part conjugated with quencher. This moderequires either that the ligand have a plurality of determinant orepitopic sites, or alternatively, that where the ligand has only one ortwo epitopic sites, a poly(ligand analog) be prepared. That is, theligand can only accommodate a few, usually from about 1 to 2 antibodiessimultaneously. As previously indicated, poly(ligand analog) is preparedby conjugating ligand analog to a nucleus molecule of high molecularweight.

In the assay where the ligand is covalently conjugated to chromophore,the assay response in going from no ligand to increasing concentrationsof ligand is a smooth curve with increasing fluorescence, until themaximum amount of fluorescence is obtained. A similar result is observedwhen one employs poly(ligand analog) to measure ligand andreceptor-fluorescer and receptor-quencher. However, with an antigen,which has a plurality of determinant sites and only receptor-quencherand receptor-fluorescer are added to the unknown to be assayed, at zeroantigen concentration, there is a maximum fluorescence which diminisheswith increasing antigen concentration to reach a minimum and thenincreases again to maximum fluorescence.

The cause of this biphasic result is straight-forward. As antigen isadded, quencher and fluorescer are brought together on the surface ofthe antigen, so that some quenching occurs. With increasing antigenconcentration, more and more of the two receptors are brought togetherat the surface of the antigen with increasing quenching. However, atsome concentration, quenching reaches a maximum (fluorescence reaches aminimum). With increasing antigen, the amount of receptor bound to anyone antigen diminishes so that the amount of quenching also diminishes.Finally, at high concentrations of antigen, the amount of receptor boundto any one antigen is insufficient to provide quenching. Therefore, whenassaying for antigen, it may be necessary to carry out the assay at twodifferent dilutions of the antigen. In this way one can determinewhether one is on the declining portion or increasing portion of thecurve.

The concentration of poly(ligand analog), based on available ligandanalog, will fall within the same ranges indicated for the ligandcovalently bound to chromophore.

In carrying out the assay with the two conjugated receptors, e.g.antibodies, the antigen is combined with the antibodies usually in thepresence of about 0.1 to 1 mg/ml of a protein, e.g. albumin, andincubated for a sufficient time, generally from about 5 sec to 6 hours,more usually from about 1/2 min to 2 hours, preferably one to 30 min, ata temperature in the range of about 15° to 40° C. The considerationsdetermining the time for incubation have been discussed previously.

With poly(ligand analog), the two conjugated antibodies are combinedwith the unknown to be assayed, incubated, and the poly(ligand analog)added and the mixture further incubated. The times and temperaturespreviously indicated are also applicable in this assay.

The sample is then introduced into a fluorometer, and the fluorescencedetermined upon exciting with light of the appropriate wavelength. Thefluorescence may be from the fluorescer or quencher depending upon thewavelength band measured. The assay can be carried out manually or beautomated.

The subject method is readily adaptable to determine the presence ofantibodies or antigens in human physiological fluid using a two stepmethod and receptor-chromophores (Ch₁ and Ch₂) for gamma globulin, e.g.human. One can readily differentiate by the difference in molecularweight between the aggregation of antibodies or other receptor moleculeswhich are bound to an antigen and the antibodies or other receptorswhich are free in solution.

Depending on whether one wishes to determine the presence or absence ofan antigen or antibodies in a human physiological fluid, e.g. blood, onewould add the complementary material, usually in substantial excess tothe maximum concentration of interest. For example, if one wished todetermine the presence of antibodies in serum to a particular antigen,one would add to antigen to the physiological fluid, where the antigenis bonded to an insoluble matrix or a high molecular weight polymer, andthen separate the bound from unbound antibodies, for example, bycentrifugation. After separating the precipitate from the supernatant,the precipitate is redispersed and assayed in accordance with theinvention for the presence of human gamma globulin. Only in the presenceof antigen will human gamma globulin be present in the precipitate.Therefore, the presence of human gamma globulin in the precipitateindicates the presence of antibodies t the antigen in the serum.

The two step metod can be used for determining a wide variety ofantigens and antibodies using the same receptor-chromophores. The methodprovides a direct determination of antibodies to specific antigens.Antigens can be indirectly determined by adding antibodies to the fluidsuspected of containing the antigen and then assaying for the presenceof antibodies in the precipitate after separation of bound and unboundantibodies.

The double receptor technique, where anti-ligand andanti-(anti-ligand)-chromophore are employed, is a homogeneous techniquewhich allows for the determination of haptens, antigens, andanti-ligand, particularly where the ligand is a polyepitopic antigen.

In one mode, for detection of a ligand, ligand analog is conjugated to achromophore, particularly fluorescer. The other chromophore,particularly quencher, is conjugated to anti(anti-ligand) to provideanti(anti-ligand)-chromophore, which is employed in conjunction withanti-ligand as a receptor composition for ligand. In this manner, onecan bind a larger number of quencher molecules to the ligand, enhancingthe opportunity for quenching. In effect, the anti-ligand provides forincreasing the number of quencher molecules capable of being bound tothe ligand.

The concentrations of the reagents will parallel the analogous reagentsfor the single receptor technique with the anti(anti-ligand)-chromophorebeing in molar excess to the anti-ligand, generally the mole ratio beingfrom about 1.5 to 10:1. If desired, individual F_(ab) units can beemployed rather than intact IgG.

The next mode has both chromophores indirectly bound to ligand. In thismode, only anti-ligand and anti(anti-ligand)-Ch₁ andanti(anti-ligand)-Ch₂ are employed. However, prior to introduction ofthese reagents, a portion of the anti-ligand will be combined withanti(anti-ligand)-Ch₁ and another portion with anti(anti-ligand)-Ch₂, soas to become bound. Desirably, the anti(anti-ligand) will bemonofunctional, e.g. F_(ab). The anti(anti-ligand)-Ch₁ and -Ch₂ bound toanti-ligand provides comparable reagent to receptor-Ch₁ and receptor-Ch₂respectively. Similar ratios of anti(anti-ligand)-chromophores toanti-ligand may be employed as previously indicated.

In a preferred embodiment, anti-ligand from two different species, e.g.mammalian species, are employed, for example, sheep and cows. In thissituation, the epitopic or haptenic sites are different for the twoanti-ligands for the same ligand. In referring to anti-ligand from twodifferent sources, anti-ligand will be preceded by a small letter, e.g.a-(anti-ligand). In this mode, the anti-ligand andanti(anti-ligand)-chromophore need not be precombined. The ratios of thevarious reagents would parallel the analogous reagents in the previouslydescribed assays.

The chromophore reagents would be anti(a-anti-ligand)-Ch₁ andanti(b-anti-ligand)-Ch₂. Thus, Ch₁ would be associated with onlya-(anti-ligand) and Ch₂ with b-(anti-ligand).

This technique allows for the determination of assemblages in solution,where members of the assemblage differ by at least one epitopic site.One can prepare a-(anti-ligand) for one member of the assemblage andb-(anti-ligand) for another member of the assemblage. Quencher andfluorescer would be brought together only when the two members are boundtogether.

Using anti-ligand from two different sources can also be employed withadvantage with a ligand to avoid having to precombine anti-ligand withthe anti(anti-ligand)-chromophore. There is also the additionalversatility of being able to follow the combining of two compounds, e.g.as in a chemical reaction or an association, or the division of onecompound into two separate entities, e.g. disassociation. In this mode,the anti-ligands from the two species would each be concerned withdifferent portions of the molecule.

The reagents can be provided in separate vials or mixed in a drylyophilized state or an aqueous, normally buffered (pH 5-10; usually6.5-8.5) solution of any desired concentration. Preferably,anti(a-anti-ligand) would not be combined with a-(anti-ligand) insolution as a reagent for a long period prior to use. Conveniently, thetwo anti-ligands could be combined and the two anti(anti-ligand)s.

A particular advantage of using the double receptor is that the samepair of (anti(anti-ligand)-chromophore)s can be employed irrespective ofthe ligand, only the pairs of anti-ligand varying with the ligand.

For determining the presence of antibodies to a particular antigen, onewould carry out the assay as if one was determining the antigen, exceptthat a known amount of antigen would be added to the assay medium. Anyantibody present in the unknown would act to diminish the amount of theanti(anti-ligand)-chromophore bound to the antigen and thus diminish theamount of quenching which would occur in the absence of antibody. Ofcourse, the anti-ligand would be from different species (other thanmammalian) than the antibody to be determined.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

(All temperatures not otherwise indicated are in Centigrade. All partsnot otherwise indicated are parts by weight. All buffer solutions areaqueous buffer. All symbols not otherwise defined are intended to havetheir normal meaning.)

The following symbols are employed:

IgG--gamma-globulin;

IgG(x)--anti-x;

R--tetramethylrhodamine, e.g. RIgG(x) tetramethylrhodamine conjugated toanti-x;

F--fluorescein, e.g. (FIgG(x) fluorescein conjugated to anti-x; and

hIgG--human gamma-globulin. EXAMPLE I

Fluorescein Isothiocyanate (FITC) Conjugate to O³ -aminoethylmorphine(FLUMO'S')

A. Fluorescein amine (0.5 g) (Sigma, isomer I, pure, tlc MeOH/CHCl₃ 1:3)was dissolved in 20 ml of dry acetone (dried on anh. K₂ CO₃) and addeddropwise at room temperature to 3 ml of thiophosgene in 5 ml of acetonewith strong stirring (1/2 hr). Stirring was continued for 1 hour and theresulting precipitate, cooled with an ice-bath to 5°, was rapidlyfiltered through a fine sintered glass funnel. The precipitate waswashed with dry acetone (3 ml) and then with 5×5 ml 6 N HCl whilecrushing with a spatula until it all turned deep red, followed by dryingin vacuo (80° KOH) overnight. The isothiocyanate obtained was pure (tlc50% MeOH/DMF).

B. O³ -aminoethylmorphine (100 mg) is dissolved in 5 ml of acetone andadded to a mixture of acetone (20 ml), water (5 ml), and triethylamine(0.07 ml). To this solution is added a solution of FITC (100 mg) inacetone (5 ml) dropwise with stirring during 15 min. Stirring iscontinued for an additional 80 min, while adjusting the pH of thereaction mixture to 9.5 with drops of dilute triethylamine solution inacetone (1.4 ml/10 ml acetone). The acetone is then partially removedwith a rotary evaporator at room temperature. The product is thenprecipitated by bubbling CO₂ through the solution with simultaneousaddition of H₂ O (up to 10 ml) until the pH drops to 6-6.5. Theprecipitate is rapidly filtered on a sintered glass funnel and washedwith H₂ CO₃ solution (2 ml, pH 6.0). Yield 60 mg. The filtrate andwashings are combined and a second crop is obtained by repeating thebubbling of CO₂ as described. Yield 27 mg. The product is driedovernight under vacuum at 80° over P₂ O₅. Total 87 mg. The product showsa single spot on tlc (50% methanol in dimethylformamide), Rf=0.45.

EXAMPLE II Purification and Labeling or Morphine Antibody (IgG(m)) withTetramethylrhodamine Isothiocyanate (TRITC)

A.

(a) Preparation of Morphine--Immunoadsorbent

Cyanogen bromide activated Sepharose 4B coupled withhexamethylenediamine (8-10μ mole/1 ml packed gel) was prepared accordingto the company's directions (Pharmacia, Upsala). Wet gel (2.5 ml) wassuspended in borate buffer (10 ml, 0.1 N, pH 8.8), the mixed anhydrideof O³ -carboxymethylmorphine and isobutyl chloroformate (0.1 mmole,large excess) in DMF (2 ml) added in the cold (0°), and the mixtureallowed to react for 3 hours. The gel was filtered and washedsuccessively with H₂ O (500 ml), 0.1 M borate buffer pH 9.0 (500 ml), H₂O (500 ml), dilute HCl+0.1 M NaCl, pH 2.5 (1500 ml), and H₂ O (1000 ml).No morphine could be detected at the end of the washings. The estimationof bound morphine was carried out by a dilute acetic acid hydrolysismethod (Failla, et al., Anal. Biochem., 52, 363 (1973). The uv spectrumwas compared to that of O³ -carboxymethylmorphine. The bound morphineequivalent was 5.05μ mole/1 ml packed gel.

(b) Purification of Morphine Antibody

The morphine Sepharose conjugate (2.5 ml) was packed in a 1/4" o.d.column and washed successively with 100 ml of borate buffer 0.1 M pH9.0, H₂ O, dilute HCl pH 1.5, H₂ O, and the same borate buffer. Stocksheep IgG solution (7 ml, 2.18×10⁻⁴ M binding sites) was applied to thecolumn followed by washing with borate buffer 0.1 M pH 9.0, until noprotein could be detected in the effluent (uv). All the antimorphineactivity was retained by the column as determined by morphine spin-labelmeasurement. (See U.S. Pat. No. 3,690,834). Washing was continued withglycine-HCl buffer 0.1 N pH 4.0 whereby no protein was eluted. Antibodywas then eluted with glycine-HCl buffer 0.1 N pH 1.5 and 3 ml fractionswere collected at room temperature in tubes containing 1 ml of 1 Nborate buffer pH 9.0. Almost all of the antibody was collected in threefractions which were combined and dialyzed for 24 hours against 0.1 Nphosphate buffer pH 7.5 (2×2000 ml). The antimorphine activity of theisolated fraction was determined with morphine spin-label and accountedfor 70% of the initially bound antimorphine activity. This fraction was100% pure as determined by the antimorphine activity titer valuecompared to protein content estimated from the uv spectrum at 280 nm.

B.

(a) Purification of Morphine Antibody-Sephadex Chromatography

The antimorphine IgG(m) solution (2 ml, ˜50 mg/ml total protein) wasseparated on Sephadex G-200 column (2×30 cm) with 0.01 M PBS (phosphatebuffered saline) pH 7.4 (flow rate 1 ml/10 min). The IgG clearlyseparated from the IgM and albumin and fractions of 2-3 ml werecollected. The obtained IgG showed no albumin on cellulose acetateelectrophoresis (Tris-barb. buffer, pH 8.8, η=0.1) and was 32-35%anti-morphine-rich IgG. Recovery depended on the cut-width of the IgGpeak collected and was usually 50% of total anti-morphine activityapplied to the column. The collected IgG fraction was dialyzed against0.01 M phosphate buffer pH 7.5.

(b) Bovine Serum Albumin (BSA)-Immunoadsorbent Treatment

BSA was coupled with CNBr activated Sepharose 4B (Pharmacia) accordingto the company's instructions (50% excess of BSA was used over therecommended amount). Five ml of the Sephadex chromatographed IgGsolution (20 mg/ml) were applied to the BSA-immunoadsorbent column (1×15cm) and run through with 0.01 M phosphate buffer pH 7.5. The collectedprotein can out in 20 ml and was assayed for protein content (uv) andantimorphine activity (spin-label method). Recovery of protein was 70%and recovery of antimorphine activity was 90-92%.

(c) Antimorphine (IgG(m)) Labeled with TRITC (RIgG(m))

To a solution of IgG(m) (7 mg/0.5 ml) in 0.01 M phosphate buffer pH 7.5is added crystalline potassium carbonate up to pH 10.0-10.5 withstirring at room temperature. TRITC (tetramethylrhodamineisothiocyanate) (15-1000 μg) dissolved in acetone (3-30 μl) is thenadded and stirring is continued for 3 hrs. Initially the pH drops to 9.0and then stays stable, and is maintained at 9.0-9.5 if necessary, bycareful addition of crystalline potassium carbonate. The reactionmixture is then applied to a Sephadex G-25(M) column (1×15 cm) with 0.01M phosphate buffer pH 7.5 and elution of the first colored band whichseparates completely from other bands is collected in 10-15 min. Theseparation is repeated twice in order to ensure complete removal of freedye. In case of formation of a precipitate, the precipitate is removedby centrifugation prior to the separation on Sephadex. The followingtable describes the preparation of conjugates with various degrees oflabeling by the above procedure:

    ______________________________________                                        Protein Concen-                                                               (% Anti-                                                                              tration  Dye (TRITC)                                                                              D/P*   % Activity                                 morphine)                                                                             mg/0.5ml μg      (M/M)  Recovered                                  ______________________________________                                        IgG (45)                                                                              7.1       15        0.9    86                                         IgG (45)                                                                              7.1       50        2.2    89                                         IgG (45)                                                                              7.1      150        4.4    75                                         IgG (45)                                                                              7.1      400        15-16  75                                         IgG (45)                                                                              7.1      750        20-23  70                                         ______________________________________                                         *D/P = Dye/Protein                                                       

EXAMPLE III Fluorescein Isothiocyanate (FITC)-Labeled Morphine Antibody(FIgG(m))

(a) Conjugation Procedure

Four 1 ml fractions of affinity chromatographed morphine antibody (3.06mg protein/ml) (See Example II) in 0.01 M phosphate buffer pH 7.5, werebrought to pH 9.5 with crystalline sodium carbonate (Na₂ CO₃). 10, 20,30, and 50 μl of an acetone solution of FITC (2 mg/300 μl) were addedrespectively to the four antibody fractions at room temperature withstirring. After 3 hrs, the four reaction mixtures were combined, thendivided into 8 equal portions and each passed through Sephadex G-25column (1×15 cm) equilibrated with 0.01 M phosphate buffer pH 7.5.Elution with the same buffer yielded (the first colored band) theconjugate which was free of unreacted dye.

(b) Separation of FITC Conjugate on DEAE-Cellulose Column

(See M. Goldmand in "Fluorescent Antibody Methods," Academic Press ed.,1968, pp. 104-107). The FITC-antimorphine conjugate was applied to aDEAE-cellulose column (1×3 cm) equilibrated with 0.01 M phosphate bufferpH 7.3. Elution with the same buffer and with increasing NaClconcentration yielded fractions of increasing dye content. The dyecontent D/P of the various fractions was determined with the Wells'nomograph (A. F. Wells, C. E. Miller and M. K. Nadel, Appl. Microbiol,14, 271 (1966). The antimorphine activity was determined as usual withmorphine spin-label. The following fractions were obtained:

    ______________________________________                                        Fraction    Protein       D/P                                                 No.         mg            mole/mole                                           ______________________________________                                        1           1.75          1.5                                                 2           1.3           3.0                                                 3           1.15          6.0                                                 4           1.42          9.0                                                 ______________________________________                                    

EXAMPLE IV Purification of Antibody to Human Gamma-Glubulin (IgG(hIgG))and Conjugation with FITC (FIgG(hIgG)) and TRITC (RIgG(hIgG))

(a) Purification of Antibody to Human IgG by Affinity Chromatography

Sepharose-4B (2 g) was coupled with 18 mg human gamma-globulin (hIgG) asdescribed in the company manual (Pharmacia, Upsala). Rabbit antiserum(50 ml) to hIgG (5 mg antibody/ml) (IgG(hIgG)) was obtained fromAntibodies Incorporated. A column (1×3 cm) of the above Sepharose-hIgGconjugate was prepared with 0.01 M borate buffer pH 8.0. The antiserumwas passed through the column, followed by washing with the same bufferuntil no protein could be detected in the eluent. The column was furtherwashed with 0.1 M glycine.HCl buffer pH 5.0. The antibody was theneluted with 0.1 M glycine.HCl buffer pH 2.5; fractions of 3 ml werecollected and immediately neutralized with 0.5 M borate buffer 9.0. Thetotal volume of antibody solution thus collected was 30 ml. The antibodysolution was dialyzed overnight against 0.05 M phosphate buffer pH 8.0,then concentrated with Aquacide and dialyzed again. The final volume of11 ml and the protein-antibody content 3.76 mg/ml as determined from theabsorption spectrum at 280 nm. Antibody recovered was 83%.

(b) Preparation of FIgG(hIgG)

(i) The above antibody solution (1 ml) in 0.05 M phosphate buffer pH 8.0was brought to pH 9.5 with crystalline Na₂ CO₃. FITC (100 μg) in 10 μlof acetone was added at room temperature and stirred for 3 hrs. Theconjugate was then separated on Sephadex G-25(M) (1×10 cm) equilibratedwith 0.05 M phosphate buffer pH 8.0. The conjugate was collected in 1.5ml; it had D/P=4.3 (M/M) (dye/protein) and 2.05 mg/ml as determined withthe Wells' nomograph.

(c) Preparation of RIgG(hIgG)

(i) The above described antibody solution (1 ml) in 0.05 M phosphatebuffer 8.0 was brought to pH 9.5 with crystalline Na₂ CO₃. TRITC (0.5mg) in acetone (20-30 μl) was added at room temperature and the mixturestirred for 3 hrs. A precipitate formed which was removed bycentrifugation and discarded. The conjugate was then separated twice onSephadex G-25 column (1×10 cm) equilibrated with 0.05 M phosphate bufferpH 8.0. The product was recovered in a 2 ml volume and had D/P=10 and0.7 mg/ml as determined from the absorption spectrum at 280 and 516 nm.

(ii) DEAE-cellulose separated IgG fraction (27.6 mg/ml) of Rabbitantiserum to hIgG (6.4 mg antibody/ml) was obtained from Antibodies Inc.The above protein solution (0.5 ml) was brought to pH 9.5 withcrystalline Na₂ CO₃, and 3 mg of TRITC in 50 μl of acetone+0.5 ml H₂ Owere added with stirring in the cold (4°). After 3 hrs, a precipitateoccurred and was filtered off. The resulting violet solution wasseparated successively twice on Sephadex G-25(M) column (2×30 cm)equilibrated with 0.05 M phosphate buffer pH 8.0. The resultingconjugate was 0.1 mg antibody/ml and had D/P=12-15 (M/M) as calculatedfrom the absorption spectrum.

EXAMPLE V Conjugation of Human Gamma-Globulin (hIgG) to Fluorescein(FhIgG)

One mg of HIgG (Human IgG) dissolved in 0.4 ml of 0.1 M phosphate bufferpH 7.5, was brought to pH 9.5 with crystalline Na₂ CO₃. A solution (10μl) of FITC (70 μg) in acetone was added with stirring and mixed for 3hrs at room temperature. The resulting solution was separated two timeson Sephadex G-25 (M) column (1×15 cm) equilibrated with 0.05 M phosphatebuffer pH 8.0. The eluted FITC-hIgG conjugate solution was 0.58 mg/ml inconcentration and had D/P=5.5 (M/M) as determined by the Wells'nomograph.

EXAMPLE VI Morphine Conjugated to Bovine Serum Albumin (BSA-44 m)

O³ -Carboxymethyl morphine (3.43 g) and 1.31 ml isobutyl chloroformatewere combined in 30 ml DMF at 0°. The resulting clear solution was thenadded to a stirring solution of 2.88 g BSA and 13 g NaHCO₃ in 600 ml ofwater at 0°. Addition was carried out by means of a syringe with its tipbelow the solution surface. The solution was stirred in a cold roomovernight.

After passing the solution through a large Sephadex column, the effluentwas concentrated to 60 ml with Dow HFD/1 overnight and lyophilized toyield 3.1 g. By uv analysis the product was shown to have an average ofabout 44 morphine groups.

In order to demonstrate the effectiveness of the subject assays usingquenching of fluorescence as a method of measuring the presence of aligand, a number of different assays were carried out employingdifferent protocols.

The first assay to be considered is the assay for morphine and codeineemploying the fluorescein isothiocyanate conjugate to O³-aminoethylmorphine (FLUMO'S').

As a first part of this assay, a number of antibody conjugates havingvarying degrees of labeling of rhodamine were combined with FLUMO'S' todetermine the maximal quenching. The FLUMO'S' was at a concentration of1.83×10⁻⁹ M in 0.05 M borate buffer pH 8.0. Fluorescence relativeintensity at F_(max) =516-518 nm was recorded by scanning from 490 nm to530 nm, excitation line was 462-464 nm and slits were adjusted with asensitivity knob to keep the peak on scale with a Perkin-Elmer ModelMPF-2A fluorescence spectrophotometer. The spectrophotometer cell, 1 cmpath length (3 ml in volume), was installed in a two mirrorcombination-base. The conjugated antibody was allowed to incubate withFLUMO'S' at room temperature in pyrex vials for 30-40 min before takingthe fluorescence reading.

The dye/protein ratio (D/P) (M/M) for the conjugates was 0.9, 2.2, 4.4,15-16, and 20-22. The results reported for relative efficiency (1/2 ofthe maximum quenching in % divided by the corresponding number ofbinding site equivalents) were respectively 6, 16.4, 24, 51.4, and 31.5.

In carrying out the assay, the following reagents were employed:FLUMO'S'--1.38×10⁻⁷ M; RIgG(m) D/P 30, 4.58×10⁻⁷ M; borate buffer 0.05 MpH 8.0; standard morphine solutions (1.5×10⁻³ -1.5×10⁻⁷ M). Incubationwas in glass tubes.

Procedure: equal amounts of RIgG(m) (40 μl) were diluted with 0.05 Mborate buffer, pH 8.0 (2940-2990 μ1) and incubated at room temperaturewith increasing amounts of morphine (5-10 μl of the standard morphinesolutions) for one hour. FLUMO'S' (10 μl) was then added and the mixtureincubated for an additional one hour. The final volume of each tube was3 ml. The final concentration of FLUMO'S' was 4.6×10⁻¹⁰ M and that ofRIgG(m) 6.1×10⁻⁹ M in binding sites. The results are reported in thefollowing table as fluorescence intensity increase as percent of maximumfluorescence possible (FLUMO'S' without quenching antibody).

                  TABLE I                                                         ______________________________________                                        morphine     signal                                                           (molarity)   intensity    % of F.sub.max                                      ______________________________________                                          0          27            33.33                                              2.5 × 10.sup.-9                                                                      28           34.5                                                5 × 10.sup.-9                                                                         29.5        36.4                                                2.5 × 10.sup.-8                                                                      35           43.2                                                5 × 10.sup.-8                                                                        38           46.9                                                2.5 × 10.sup.-7                                                                      54           66.6                                                5 × 10.sup.-7                                                                        60           74                                                  2.5 × 10.sup.-6                                                                      74           91.3                                                5 × 10.sup.-6                                                                        78           96.3                                                ______________________________________                                    

The study was repeated except that codeine was employed in place ofmorphine. The following table indicates the results.

                  TABLE II                                                        ______________________________________                                        Codeine      signal                                                           (molarity)   intensity    % of F.sub.max                                      ______________________________________                                          0          27           32.9                                                2.5 × 10.sup.-9                                                                       30.5        37.2                                                5 × 10.sup.-9                                                                        36           43.9                                                2.5 × 10.sup.-8                                                                      51           62.2                                                5 × 10.sup.-8                                                                        58           70.7                                                5 × 10.sup.-7                                                                        75           91.5                                                2.5 × 10.sup.-6                                                                      80           97.5                                                ______________________________________                                    

The assay was repeated, but instead of the rhodamine labeled morphineantibody (RIgG(m)) having a D/P (dye/protein) (M/M) ratio of 30, RIgG(m)was employed having a D/P of 22. The following are the results employingmorphine.

                  TABLE III                                                       ______________________________________                                        morphine     signal                                                           (molarity)   intensity    % of F.sub.max                                      ______________________________________                                          0          24.5         29.9                                                2.5 × 10.sup.-10                                                                     26.0         31.7                                                5 × 10.sup.-10                                                                       26.5         32.3                                                2.5 × 10.sup.-9                                                                      28.0         34.1                                                5 × 10.sup.-9                                                                        29.0         35.4                                                1 × 10.sup.-8                                                                        33.5         40.8                                                2.5 × 10.sup.-8                                                                      40.0         48.8                                                5 × 10.sup.-8                                                                        45.5         55.5                                                1 × 10.sup.-7                                                                        53.0         64.6                                                2.5 × 10.sup.-7                                                                      63.0         76.8                                                5 × 10.sup.-7                                                                        68.0         82.9                                                1 × 10.sup.-6                                                                        72.5         88.4                                                2.5 × 10.sup.-6                                                                      80.0         97.5                                                5 × 10.sup.-6                                                                        82.0         100                                                 ______________________________________                                    

The next study which was carried out employed a polyligand, namely,morphine conjugated to bovine serum albumin, having an average number of44 morphines per albumin. In a first test, the polyligand was employedas a synthetic protein in that the polyligand has a plurality ofmorphine epitopic sites. In a second series of tests, the polyligand wasemployed in an assay for morphine or codeine. In both these assays,neither chromophore is covalently bound to the epitopic site ofinterest, but rather each becomes bound through antibody. Thus, there isa random binding of antibody to morphine on the polyligand. At theconcentrations of interest, in a study not described here, it was foundthat optimum quenching was obtained where a ratio of quencher asreceptor- quencher to fluorescer as receptor-fluorescer was about 5 to1.

In the first test, which is an assay for the poly(ligand analog), aseries of tubes were prepared each containing 6.4×10⁻⁹ M (in bindingsites) of antimorphine having a D/P ratio of fluorescein/antibody of 9and 3.47×10⁻⁸ M (in binding sites) of antimorphine having a D/P ratio ofrhodamine/antibody of about 22 in 0.05 M phosphate buffer, pH 8.0,containing 1.2×10⁻⁶ M bovine gamma-globulin. Various amounts of themorphine conjugated bovine serum albumin (approximately 44 morphines peralbumin) (0.012-1.2 μg) were added (in 5-10 μl) to each of the tubes sothat the final volume was 0.5 ml and incubated at room temperature for30 min. The fluorescence of each of the tubes is then measured andexpressed in percentage of maximal fluorescence possible (when nomorphine conjugated BSA is present). The results are in the followingtable.

                  TABLE IV                                                        ______________________________________                                        44m-BSA                                                                       μg (added)    % of F.sub.max                                               ______________________________________                                        0                100                                                          0.012             84.5                                                        0.024            72                                                           0.048             64.5                                                        0.084            61                                                           0.12             66                                                           0.24             74                                                           0.48             83                                                           1.20             92                                                           ______________________________________                                    

For assaying for codeine, the following procedure was employed.Employing the same antimorphine-fluorescein (FIgG(m)) andantimorphine-rhodamine (RIgG(m)) as employed above, 30 μl of the FIgG(m)(2.64×10⁻⁷ M) and 30 μl of the RIgG(m) (1.44×10⁻⁶ M) were diluted in aseries of tubes with 0.05 M phosphate buffer, pH 8.0, containing1.5×10⁻⁶ M bovine gamma-globulin (390-430 μl) Codeine in increasingconcentrations (1.5×10⁻³ -1.5×10⁻⁶ M) is then added (10-40 μl) and themixture incubated at room temperature for 0.5 hr. To each of the tubesis then added 10 μl (0.24 μg) of the morphine-bovine serum albuminconjugate used previously and the tubes incubated for an additional onehour. The final volume in each tube was 0.5 ml. The fluorescence of eachof the tubes at 518 nm was then recorded and expressed as percentage ofmaximal fluorescence possible (when no morphine-BSA conjugate ispresent). The following table indicates the results.

                  TABLE V                                                         ______________________________________                                        Codeine                                                                        (M)           % of F.sub.max                                                 ______________________________________                                          0            49                                                             3 × 10.sup.-9                                                                          51                                                             6 × 10.sup.-9                                                                          54                                                             1.2 × 10.sup.-8                                                                         56.5                                                          3 × 10.sup.-8                                                                          62                                                             6 × 10.sup.-8                                                                           70.5                                                          1.2 × 10.sup.-7                                                                        81                                                             3 × 10.sup.-7                                                                           90.5                                                          6 × 10.sup.-7                                                                           94.5                                                          1.2 × 10.sup.-6                                                                        100                                                            3 × 10.sup.-6                                                                          100                                                            ______________________________________                                    

The next two studies involve the natural protein human gamma-globulin.In the first study, human gamma-globulin-fluorescein (FhIgG) D/P 5.5 wasemployed for the determination of human gamma-globulin. A series oftubes were prepared, each containing 100 μl of 0.017 mg/ml of anti-humangamma-globulin-rhodamine conjugate (RIgG(hIgG) D/P 12-15 in 0.05 Mphosphate buffer, pH 8.0 (330-380 μl) containing BSA (0.6 mg/ml).Increasing amounts of human gamma-globulin (in 15-35 μl) were then addedand incubated for 30 min at room temperature. To the solutions was thenadded 30 μl of 0.014 mg/ml of FhIgG. The final volume in each case was0.5 ml. The final concentration of the FhIgG was 5.4×10⁻⁹ M, while theconcentrations of human gamma-globulin ranged from 4.84×10⁻¹⁰ to6.45×10⁻⁸ M. After a second incubation period of 30 min, thefluorescence of the tubes at 522 nm was recorded as percentage ofmaximal fluorescence possible. The following table indicates theresults.

                  TABLE VI                                                        ______________________________________                                        HIgG                                                                          (M)            % of F.sub.max                                                 ______________________________________                                          0            28                                                             4.84 × 10.sup.-10                                                                      33                                                             8.06 × 10.sup.-10                                                                      36                                                             1.13 × 10.sup.-9                                                                       38                                                             1.61 × 10.sup.-9                                                                       46                                                             3.22 × 10.sup.-9                                                                       68                                                             4.84 × 10.sup.-9                                                                       81                                                             8.06 × 10.sup.-9                                                                       91                                                             1.13 × 10.sup.-8                                                                       93                                                             1.61 × 10.sup.-8                                                                        95.5                                                          3.22 × 10.sup.-8                                                                       96                                                             6.45 × 10.sup.-8                                                                       98                                                             ______________________________________                                    

In the next determination, the human gamma-globulin was assayed byemploying anti-human gamma-globulin-fluorescein (FIgG(hIgG)) conjugateand anti-human gamma-globulin-rhodamine conjugate (RIgG(hIgG)). Thefluorescein conjugate had a D/P of 4.3 and the rhodamine conjugate had aD/P of 10. All reagents were diluted with 0.05 M phosphate buffer, pH8.0, containing 0.6 mg/ml of bovine serum albumin. A series of tubeswere prepared, each containing 400 μl of the indicated buffer. To eachof the tubes was added, 30 μl of 2.7 μg/ml of FIgG(hIgG) and 30 μl of 35μg/ml of RIgG(hIgG). The tubes were mixed and increasing amounts ofhuman gamma-globulin added in 40 μl solutions and incubated at roomtemperature for one hour. The fluorescence of the tubes was thenmeasured and expressed as percent of total fluorescence in the absenceof human gamma-globulin. The following table indicates the results.

                  TABLE VII                                                       ______________________________________                                        Human IgG                                                                       (M)          % of F.sub.max                                                 ______________________________________                                        3 × 10.sup.-11                                                                         100                                                            6 × 10.sup.-11                                                                         95.7                                                           1.2 × 10.sup.-10                                                                       93.2                                                           1.8 × 10.sup.-10                                                                       89.5                                                           2.4 × 10.sup.-10                                                                       86.5                                                           3 × 10.sup.-10                                                                         82.2                                                           6 × 10.sup.-10                                                                         70.5                                                           1.2 × 10.sup.-9                                                                        60.7                                                           1.8 × 10.sup.-9                                                                        62                                                             2.4 × 10.sup.-9                                                                        68.1                                                           3 × 10.sup.-9                                                                          69.3                                                           6 × 10.sup.-9                                                                          79                                                             1.2 × 10.sup.-8                                                                        87.7                                                           1.8 × 10.sup.-8                                                                        88.3                                                           2.4 × 10.sup.-8                                                                        93.8                                                           ______________________________________                                    

As is evidenced from the above Table VII, with increasing humangamma-globulin concentration, the fluorescence decreases to a minimumand then increases. Therefore, with an unknown, it would be necessary tocarry out two dilutions to determine which part of the curve wasinvolved.

The above results demonstrate the extreme sensitivity and wide range ofcapability of the subject assays. By employing thefluorescence-quenching phenomenon, one can assay directly for a widevariety of different compounds, both haptenic and antigenic. Reagentscan be employed, where the hapten or antigen is covalently bonded to thechromophore, or alternatively, where the compound of interest has aplurality of epitopic sites, mixtures of antibodies can be employed witha portion of the antibodies bonded to quencher and a portion of theantibodies bonded to fluorescer. In this situation, derivatives of theligand are not required for preparing reagents, where a naturallyoccurring receptor is available or the ligand is antigenic.

In addition, reagents can be prepared having a plurality of haptenic orantigenic molecules bonded to a nucleus molecule. Either the nucleusmolecule can be bonded to a chromophore and antibody employed which isconjugated to the other member of the fluorescer-quencher pair or themixture of antibodies indicated above employed. The assay is relativelyrapid, and depending upon the concentrations, various incubation timesare required. Furthermore, conventional fluorometers can be employedwhich are relatively inexpensive and easily read.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A composition for determining the presence oramount of a ligand comprising two chromophores, which are afluorescer-quencher pair, the amount of fluorescer within quenchingdistance of said quencher being affected by the presence or amount ofligand, wherein each chromophore is covalently bonded to an anti-ligandcapable of specific non-covalent binding to said ligand.
 2. Thecomposition of claim 1, which in addition includes one of saidchromophores covalently bonded to an antibody to said anti-ligand. 3.The composition of claim 1, wherein said ligand is a globulin.
 4. Thecomposition of claim 1, wherein said ligand is a hapten.